The normal function of the immune system is to protect the body by attacking and destroying foreign microorganisms, such as bacteria and viruses. Under normal conditions, these would be recognised as invaders and thus be rendered harmless.
Responsible for a subject's immune response is the lymphoid system; meaning the assemblage of lymphocytes, their precursors and derivatives, and all supportive cells. Under abnormal conditions or imbalance of the immune system, it fails to react and destroy the foreign microorganisms, mistakes own cells and tissues as foreign and attacks them, or is activated inappropriately in a hypersensitive manner.
An immune response to an antigenic agent, be it a foreign antigen or an auto-antigen, is generally characterised by the production of antibodies by B lymphocytes, and destruction of any cells displaying those antigens by T lymphocytes or natural killer (NK) cells. Defects in B or T lymphoid cells, however, may result in the development of immunodeficiency diseases or the impairment of the immune response function. The immune deficiency or defect may be congenital, i.e. caused by a mutation in a gene, or it may be acquired, e.g. through a viral infection or as a result of the ageing process. The thus produced defect may or may not be fatal, depending on the stage of stem cell or lymphocyte differentiation at which it occurs.
In addition to an antigen, lymphocytes need lymphokines for full activation. E.g., the T-helper cells do not proliferate unless they receive signals from macrophages, and T-cytotoxic cells as well as B-cells depend on signals from T-helper cells and probably also from macrophages for their full development. An adequate immune response needs the coordinated activities of several cell types, which means that modulating the expression or activity of lymphokines potentially has a beneficial effect on adverse immune responses.
After shocks, burns, extensive surgical procedures or traumatic injury, functions of the immune system can be over-stimulated within a systemic, non-discriminant, excessive whole-body inflammation, whereas other functions are dramatically paralysed. When the immunological host response is thus uncontrolled, the unbalanced states of cellular activation that are observed lead to consecutive clinical states. Such states can be acute-phase response, whole-body inflammation, anergy, sepsis, infection or organ failure and may finally lead to multiple organ failure. Multiple organ failure or dysfunction is the most common cause for death in intensive care units. As of today, therapy directed to prevent or improve uncontrolled immune response has not been able to dramatically alter the outcome.
Other examples for unbalanced situations are when the immune system attacks its own body. These situations differ in the origin of the antigen that triggers the attack and the mechanisms and manifestations of the attack. These reactions are often referred to as allergy, delayed-type hypersensitivity, allograft, and autoimmune reactions, respectively.
Anergy is another unbalanced condition, in which the body fails to react to an injected allergen or antigen. Anergy is thought to be the result of intercellular signalling after interaction between T-cell receptor (TCR) and peptide-presenting major histocompatibility complex (MHC) antigen in the absence of a “costimulatory” signal. This costimulatory signal is normally provided on the cell surface of antigen-presenting cells (APCs).
The immune system modulates its function both locally and systemically. E.g. delayed-type hypersensitivity can manifest itself either locally in a characteristic skin reaction, or in a systemic reaction in which large quantities of the antigen enter the bloodstream and which is characterised by fever, malaise, pains in the joints and reduction of the number of circulating lymphocytes. Inflammation is generally defined as a local response to cellular injury, whereas whole body inflammation or multiple organ failure is a systemic inflammation.
Posttraumatically, monocytes and macrophages are immediately hyperactivated to excessively release proinflammatory cytokines. This excessive release results in the development of whole body inflammation, followed in most patients by substantial paralysis of cell function. After 3 to 5 days this state is overcome with newly recruited monocytes/macrophages that probably lack the full spectrum of activity, though, because they are immature. In addition, patients can also undergo an anti-inflammatory phase (the compensatory anti-inflammatory response syndrome), and at times a mixed response with both pro-and anti-inflammatory components (the mixed antagonistic response syndrome) is observed.
The posttraumatic impact on the balance of cell-mediated immune regulation is thus primarily caused by a simultaneous attack on the monocyte/macrophage lineage and T cells, causing disintegration of the intact cell interaction. Traumatic stress not only effects the capacity of adequate and specific performance of each cell type; it also effects control capacity and modulatory surveillance that the monocyte/macrophage lineage and T cells normally posses for each other within a number of regulatory loops. This loss of regulatory function occurs instantaneously at the moment of injury.
Known inflammatory cytokines to cause the above-described initial systemic hyperinflammation are e.g. Tumour necrosis factor-a (TNF-alpha), Interleukin-1 (IL-1), IL-6, and Interferon gamma, which might act synergistically with TNF-alpha. Clinical trials aimed at down-regulating these mediators, using e.g. antibodies against endotoxin, TNF-alpha, antagonists of IL-1, or platelet activating factor, have so far been uniformly disappointing. Not only have these agents been found to have no regulating effect, they even increased mortality.
Furthermore, under stressful conditions, the monocyte/macrophage lineage's are easily triggered to produce and release prostaglandin E2 (PGE2), which is probably the most powerful endogenous immune suppressant. PGE2 is an inhibitor of T cell mitogenesis, IL-2 receptor expression and IgM antibody synthesis by B cells. Via intracellular elevation of cAMP levels, PGE2 also negatively controls the monocyte/macrophage lineage synthesis of TNFα and IL-1. PGE2 can inhibit the synthesis of TH1 cytokines (IL-2, IFNγ) but not the synthesis of IL-4 by TH2 cells. Thus PGE2 secretion may tip the balance in favour of a TH2 type response, leading to a switch in B cell production from IgM to IgG1 and IgE. Tipping the balance thus influences the development of a TH1 or a TH2-dominated response.
Traditional reagents and methods used to regulate a patient's immune response often result in unwanted side effects. For example, immunosuppressive reagents, such as cyclosporine A, azathioprine and prednisone are used to suppress the immune system of a patient with an autoimmune disease or patients receiving transplants. Such reagents, however, suppress a patient's entire immune response, thereby crippling the ability of the patient to mount an immune response against infectious agents not involved in the original disease. Due to such harmful side effects and the medical importance of immune regulation, reagents and methods to regulate specific parts of the immune system have been the subjects of study for many years.
Enamel matrix proteins, present in the enamel matrix, are most well known as precursors to enamel. Prior to cementum formation, enamel matrix proteins are deposited on the root surface at the apical end of the developing tooth-root. The deposited enamel matrix is the initiating factor for the formation of cementum. Again, the formation of cementum in itself is associated with the development of the periodontal ligament and the alveolar bone. As shown by the present inventors prior to the present invention, enamel matrix proteins can therefore promote periodontal regeneration through mimicking the natural attachment development in the tooth (Gestrelius S, Lyngstadaas SP, Hammarström L. Emdogain—periodontal regeneration based on biomimicry. Clin Oral Invest 4:120-125 (2000).
The enamel matrix is composed of a number of proteins, such as amelogenin, enamelin, tuft protein, proteases, and albumin. Amelogenins, the major constituent of the enamel matrix, are a family of hydrophobic proteins derived from a single gene by alternative splicing and controlled post secretory processing. They are highly conserved throughout vertebrate evolution and demonstrate a high overall level of sequence homology among all higher vertebrates examined (>80%). In fact, the sequences of porcine and human amelogenin gene transcript differ only in 4% of the bases. Thus, enamel matrix proteins, although of porcine origin, are considered “self” when encountered in the human body and can promote dental regeneration in humans without triggering allergic responses or other undesirable reactions.
Enamel matrix proteins and enamel matrix derivatives (EMD) have previously been described in the patent literature to be able to induce hard tissue formation (i.e. enamel formation, U.S. Pat. No. 4,672,032 (Slavkin)), binding between hard tissues (EP-B-0 337 967 and EP-B-0 263 086) and wound healing, such as of skin and mucosa (WO 99/43344).